Experiments Testing the Causes of Namibian Fairy Circles

The grasslands on the sandy soils of the eastern edge of the Namib Desert of Namibia are strikingly punctuated by millions of mostly regularly-spaced circular bare spots 2 to 10 m or more in diameter, generally with a margin of taller grasses. The causes of these so called fairy circles are unknown, but several hypotheses have been advanced. In October 2009, we set up experiments that specifically tested four hypothesized causes, and monitored these 5 times between 2009 and 2015. Grass exclusion in circles due to seepage of subterranean vapors or gases was tested by burying an impermeable barrier beneath fairy circles, but seedling density and growth did not differ from barrier-less controls. Plant germination and growth inhibition by allelochemicals or nutrient deficiencies in fairy circle soils were tested by transferring fairy circle soil to artificially cleared circles in the grassy matrix, and matrix soil to fairy circles (along with circle to circle and matrix to matrix controls). None of the transfers changed the seedling density and growth from the control reference conditions. Limitation of plant growth due to micronutrient depletion within fairy circles was tested by supplementing circles with a micronutrient mixture, but did not result in differences in plant seedling density and growth. Short-range vegetation competitive feedbacks were tested by creating artificially-cleared circles of 2 or 4 m diameter located 2 or 6 m from a natural fairy circle. The natural circles remained bare and the artificial circles revegetated. These four experiments provided evidence that fairy circles were not caused by subterranean vapors, that fairy circle soil per se did not inhibit plant growth, and that the circles were not caused by micronutrient deficiency. There was also no evidence that vegetative feedbacks affected fairy circles on a 2 to 10 m scale. Landscape-scale vegetative self-organization is discussed as a more likely cause of fairy circles.     

Localization of low-molecular-weight basic proteins in Bacillus megaterium spores by cross-linking with ultraviolet light.

Two low-molecular-weight basic proteins, termed A and B proteins, comprise about 15% of the protein of dormant spores of Bacillus megaterium. Irradiation of intact dormant spores with ultraviolet light results in covalent cross-linking of the A and B proteins to other spore macromolecules. The cross-linked A and B proteins are precipitated by ethanol and can be solubilized by treatment with deoxyribonuclease (75%) or ribonuclease (25%). Irradiation of complexes formed in vitro between deoxyribonucleic acid (DNA) or ribonucleic acid and a mixture of the low-molecular-weight basic proteins from spores also resulted in cross-linking of A and B proteins to nucleic acids. The dose-response curves for formation of covalent cross-links were similar for irradiation of both a protein-DNA complex in vitro and intact spores. However, if irradiation was carried out in vitro under conditions where DNA-protein complexes were disrupted, no covalent cross-links were formed. These data suggest that significant amounts of the low-molecular-weight basic proteins unique to bacterial spores are associated with spore DNA in vivo.     

Genetic and physical studies of recombinant plasmids formed between an R plasmid of compatibility group FI and sex factor F of HfrH.

Recombinant plasmids between an R plasmid of the FI group (R162/3) and the sex factor F or HfrH were produced after the conjugal transfer of this R plasmid into HfrH. Three types of recombinant plasmids were identified after the mating of HfrH (R162/3) with recA and rec+ recipients. One specimen of each type (pIP218, pIP222, pIP226) was studied in this report. All three recombinant plasmids carry the same genetic information for resistance to antibiotics (CSSuT) retained from R162/3. pIP218 retained all the other properties from F of HfrH: derepression for pilus synthesis, mobilization of the chromosome for the proximally transferred HfrH genes (thr, leu, proA), interference with T7 propagation, and ability to be cured by acridine orange. pIP222 retained from F of HfrH the derepression for pilus synthesis and the same polarity of chromosome transfer (thr, leu, proA), while pIP226 retained the interference with T7 propagation and acridine orange curing. Physical studies revealed that replication control and/or recovery of F and pIP218 as covalent circles of deoxyribonucleic acid are similar, and are different from R162/3. The new plasmids are more likely the result of a substitutive recombination event than a fusion. We propose genetic maps of these recombinant plasmids, showing the unequal participation of the parental plasmids in their formation.     

Granulosis Virus Deoxyribonucleic Acid: a Closed, Double-Stranded Molecule

Studies on deoxyribonucleic acid purified from the granulosis virus of Trichoplusia ni have revealed the presence of a closed, double-stranded superhelix which sediments at 95S relative to relaxed circles (74S) and linear (60S) forms. Molecular weight estimates show that this insect virus deoxyribonucleic acid has a size of 100 x 10(6) daltons.     

Covalent coupling of bilirubin to albumin.

A method for covalent coupling of bilirubin to albumin is described. Human serum albumin-bilirubin (1:1 complex) has been treated with water soluble carbodiimide in order to obtain covalent coupling of bilirubin to albumin. The reaction conditions have been varied with respect to pH, reaction time and concentration of reagent to obtain the optimal coupling. The prepared albumin-bilirubin compounds were investigated by spectrophotometry, gel filtration and gel electrophoresis to ascertain the covalent nature of the bond and to characterize the products further. Gel electrophoresis and gel filtration showed that a monomer fraction could be prepared, and this fraction was a suitable material for further studies.     

Covalent bonded Gag multimers in human immunodeficiency virus type-1 particles

The oligomerization of HIV-1 Gag and Gag-Pol proteins, which are assembled at the plasma membrane, leads to viral budding. The budding generally places the viral components under non-reducing conditions. Here the effects of non-reducing conditions on Gag structures and viral RNA protection were examined. Using different reducing conditions and SDS-PAGE, it was shown that oligomerized Gag possesses intermolecular covalent bonds under non-reducing conditions. In addition, it was demonstrated that the mature viral core contains a large amount of covalent bonded Gag multimers, as does the immature core. Viral genomic RNA becomes sensitive to ribonuclease in reducing conditions. These results suggest that, under non-reducing conditions, covalent bonded Gag multimers are formed within the viral particles and play a role in protection of the viral genome.     

靶向共价键药物的研究进展

药物与靶标的结合是启动药理作用的本源,共价键药物是以共享电子的方式来实现与靶标的结合,其中大多为抗感染、抗肿瘤以及心脑血管、神经系统和代谢类药物。简介共价键药物与非共价键药物的区别以及既往的重磅级共价键药物与靶标的结合特点,分类综述靶向共价键药物的理性设计及与靶标的结合反应。     

Detection and Characterization of Plasmids in Pseudomonas aeruginosa Strain PAO

Using a variety of techniques, it has been established that the sex factor FP2 has a density of 1.717 g/cm(3) (corresponding to a guanine plus cytosine [G + C] content of 58%) and a mean circular contour length of 28.5 +/- 0.6 mum (corresponding to a molecular weight of 59 x 10(6)). Another sex factor FP39 has a density of 1.719 g/cm(3) (corresponding to a G + C content of 60%) and a mean circular contour length of 26.5 +/- 0.5 mum (corresponding to a molecular weight of 55 x 10(6)). It appears that all, or nearly all, of the FP sex factor deoxyribonucleic acid occurs as covalent circular molecules under the conditions employed. In addition, these procedures have been used to demonstrate that strain PAO, a naturally occurring female (i.e., FP(-)) strain of Pseudomonas aeruginosa, harbors a number of cryptic plasmids having similar densities to the bulk of the cell deoxyribonucleic acid (1.726 g/cm(3)) and occurring as covalent circular molecules.     

Covalent attachment of proteins to peptidoglycan

Bacterial surface proteins are key players in host-symbiont or host-pathogen interactions. How these proteins are targeted and displayed at the cell surface are challenging issues of both fundamental and clinical relevance. While surface proteins of Gram-negative bacteria are assembled in the outer membrane, Gram-positive bacteria predominantly utilize their thick cell wall as a platform to anchor their surface proteins. This surface display involves both covalent and noncovalent interactions with either the peptidoglycan or secondary wall polymers such as teichoic acid or lipoteichoic acid. This review focuses on the role of enzymes that covalently link surface proteins to the peptidoglycan, the well-known sortases in Gram-positive bacteria, and the recently characterized l,d-transpeptidases in Gram-negative bacteria.     

Periodic conformations of deoxyribonucleic acids

The conformation of a deoxyribonucleotide unit in a deoxyribonucleic acid molecule can be defined by six angles, each of which specifies the relative orientations between two groups of atoms adjacent to a covalent bond. With the assumption that these atoms are hard spheres with fixed van der Waal radii, conformations are sought to minimize their overlapping. The additional requirement of the polymer having a periodic structure further reduces the allowable conformations.     

Covalent interaction of chloroacetic and acetic acids with cholesterol

The covalent interaction of chloroacetic acid with rat liver lipids was studied in vivo. Rats were given a single oral dose (8.75 mg/kg, 50 microCi) of 1-[14C]chloroacetic acid and sacrificed after 24 hours. Lipids extracted from the livers were separated into neutral lipids and phospholipids by solid-phase extraction using sep-pak silica cartridges. The neutral lipid fraction was further fractionated by preparative thin-layer chromatography followed by reverse-phase high-performance liquid chromatography. The fraction corresponding to the retention time of standard cholesteryl chloroacetate gave a pseudomolecular ion peak at m/z 480/482 ratio: (3:1) on ammonia chemical ionization mass spectrometry, and the fragmentation pattern was found to be similar to that of the standard sample. Under similar conditions, acetic acid resulted in the formation of cholesteryl acetate. The effect of such conjugation reactions on the cell membrane and their contribution to toxicity is presently unknown.     

Covalent modification of proteins as a threshold mechanism in development

Thresholds are a central but somewhat neglected aspect of cellular processes in development. An analysis has been made of the conditions in which different thresholds can be generated in the covalent modification of a number of target proteins when the concentration of an effector is continuously increased. It is assumed that the effector, which could represent a morphogen, activates, for example, kinases that phosphorylate the proteins. Thresholds are found when the modifying enzymes are saturated by their protein substrates, i.e. in conditions of zero-order ultrasensitivity (Goldbeter, A. & Koshland, D. E. 1981. An amplified sensitivity arising from covalent modification in biological systems. Proc. natn. Acad. Sci. U.S.A. 78, 6840-6844). Sequential thresholds can be generated when the kinase/phosphatase pairs differ either in the ratio of maximum modification rates or in the affinity of the effector for each kinase.     

葡萄糖氧化酶的有机相共价固定化

将葡萄糖氧化酶(GOD)在最适pH条件下冻干后,以戊二醛活化的壳聚糖为载体,分别在传统水相和1,4-二氧六环、乙醚、乙醇三种不同的有机相中进行共价固定化。通过比较水相固定化酶和有机相固定化酶的酶比活力、酶学性质及酶动力学参数,考察酶在有机相中的刚性特质对酶在共价固定化过程中保持酶活力的影响。结果表明,戊二醛浓度为0.1%、加酶量为80 mg/1 g载体、含水1.6%的1,4-二氧六环有机相固定化GOD与水相共价固定化GOD相比,酶比活力提高2.9倍,有效酶活回收率提高3倍;在连续使用7次后,1,4-二氧六环有机相固定化GOD的酶活力仍为相应水相固定化酶的3倍。在酶动力学参数方面,不论是表观米氏常数,最大反应速度还是转换数,1,4-二氧六环有机相固定化的GOD(Kmapp=5.63 mmol/L,Vmax=1.70μmol/(min.mgGOD),Kcat=0.304 s-1)都优于水相共价固定化GOD(Kmapp=7.33 mmol/L,Vmax=1.02μmol/(min.mg GOD),Kcat=0.221 s-1)。因此,相比于传统水相,GOD在合适的有机相中进行共价固定化可以获得具有更高酶活力和更优催化性质的固定化酶。该发现可能为酶蛋白在共价固定化时因构象改变而丢失生物活性的问题提供解决途径。     

Covalent stabilization of a small molecule-RNA complex

We demonstrate covalent bond formation between an RNA aptamer containing a cysteamine-tethered nucleobase and helix-threading peptides (HTPs) containing α-bromoacetamide N-termini. The reaction is high yielding and inhibited by a DNA strand Watson-Crick complementary to the aptamer sequence indicating covalent reaction is dependent on the high affinity HTP-binding site present in the folded aptamer. These results are important for future structural studies of HTP-RNA complexes and methods for the discovery of new high affinity analogs via covalent tethering strategies.     

Covalent binding of perfluorinated fatty acids to proteins in the plasma, liver and testes of rats.

Perfluorinated fatty acids alter hepatic lipid metabolism and are potent peroxisome proliferators in rodents. Two such perfluorinated acids, perfluorodecanoic acid (PFDA) and perfluorooctanoic acid (PFOA), were examined to determine if they covalently bind cellular proteins. PFDA and PFOA were found to covalently bind proteins when administered to rats in vivo. The liver, plasma and testes of male rats treated with [1-14C]PFDA or PFOA (9.4 mumol/kg) contained detectable levels of covalently bound 14C (0.1-0.5% of the tissue 14C content). Characterization of PFDA covalent binding to albumin in vitro showed that cysteine significantly decreased binding with no effect of methionine, suggesting protein sulfhydryl groups are involved. In cytosolic and microsomal incubation there was no effect of the addition of CoA, ATP or NADPH on the magnitude of the covalent binding of PFDA. Therefore PFDA need not be metabolically activated to form covalent adducts. Despite demonstration of covalent binding of PFDA and PFOA to proteins both in vivo and in vitro, the role of this macromolecular binding in perfluorinated fatty acid toxicity is not known.     

Covalent modification of phenylalanyl-tRNA synthetase with phenylalanine during the amino acid activation reaction catalyzed by the enzyme

Yeast phenylalanyl-tRNA synthetase (PRS) is shown to undergo autoaminoacylation with phenylalanine under in vitro amino acid activation conditions. Phenylalanyl adenylate enzyme complex yields a covalent phenylalanyl isopeptide exclusively with the beta subunit of the alpha 2 beta 2 enzyme. Contrary to previously reported cases of autoaminoacylation of aspartyl-tRNA synthetase and tryptophanyl-tRNA synthetase, the autoaminoacylation of PRS occurs under a specific set of conditions and results in the identification of only one labeled tryptic peptide on two types of high pressure liquid chromatography columns. The ability of PRS to undergo this covalent modification directly correlates with its ability to catalyze the synthesis of diadenosine 5',5"'-P1,P4-tetraphosphate from enzyme-bound phenylalanyl adenylate. Both reactions require the presence of low levels of zinc or cadmium and are inhibited by tRNAPhe or by low levels of low molecular weight thiols. Since diadenosine 5',5"'-P1,P4-tetraphosphate synthesis is known to be catalyzed in vivo in response to oxidation stress, it is also likely that the autoaminoacylation of phenylalanyl-tRNA synthetase may occur in vivo under a similar set of conditions. These reactions are thus not simply the result of accumulation of phenylalanyl adenylate and probably reflect conformational changes in the protein which are brought about by its interaction with zinc or cadmium.     

Covalent immunoglobulin labeling through a photoactivable synthetic Z domain

Traditionally, labeling of antibodies has been performed by covalent conjugation to amine or carboxyl groups. These methods are efficient but suffer from nonspecificity, since all free and available amine/carboxyl groups have the possibility to react. This drawback may lead to uncontrolled levels and locations of the labeling. Hence, the labeled molecules might behave differently and, possibly, the binding site of the antibody will also be affected. In this project, we have developed a highly stringent method for labeling of antibodies by utilizing an immunoglobulin-binding domain from protein A, the Z domain. Domain Z has been synthesized with an amino acid analogue, benzoylphenylalanine, capable of forming covalent attachment to other amino acids upon UV-exposure. This feature has been used for directed labeling of immunoglobulins and subsequent use of these in different assays.     

Effect of protein synthesis on plasmid maintenance in Streptococcus faecalis.

Plasmid-to-chromosome ratios in Enterobacteriaceae, upon interruption of protein synthesis by chloramphenicol, are either conserved or increased when measured by dye buoyant density centrifugation. We have found, on the other hand, that the effect of inhibition of protein synthesis on the amount of covalently closed circular deoxyribonucleic acid visualized by this method in two strains of Streptococcus faecalis appears to differ from these established systems. A three- to sixfold decrease in covalently closed circular deoxyribonucleic acid was observed when lysates of chloramphenicol-treated cultures were submitted to dye buoyant density centrifugation. A loss of covalently closed circular deoxyribonucleic acid was also evident from electrophoretic profiles of these lysates. Several conditions which could account for the apparent loss of covalently closed circular deoxyribonucleic acid upon inhibition of protein synthesis are discussed.     

Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II

Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol:fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation.